Abstract: TMPRSS13 is a cell-surface anchored serine protease that is up-regulated in human breast carcinoma cancer cells compared to normal breast epithelial cells. Using a TMPRSS13 deficient mouse model, we found that TMPRSS13 plays a causal role in mammary carcinogenesis, where it contributes significantly to primary tumor initiation, growth, and metastasis to the lungs. In cultured human breast cancer cells, silencing of TMPRSS13 causes decreased proliferation and increased apoptosis. The overarching goal is to validate TMPRSS13 as a potential new therapeutic target in breast cancer. We will perform both functional and mechanistic experiments to pinpoint the role of TMPRSS13 in breast cancer using parallel and complimentary hypothesis-driven experiments and unbiased approaches. The central hypothesis to be tested is that TMPRSS13 promotes tumor progression by activating pro- survival and pro-invasive signaling and represents a novel target for breast cancer treatment. We formulated three independent specific aims to test this hypothesis. In Aim 1, we will determine how TMPRSS13 promotes pro-survival and invasion. We discovered that two central regulators of epithelial-to-mesenchymal transition (EMT), and members of the Snail zinc-finger transcription factor family, Snail and Slug, are regulated by TMPRSS13. The significance of this finding and the functional relationship between TMPRSS13 and Snail/Slug will be tested in a variety of cellular assays. In parallel, state-of-the-art RNA-Seq analysis and quantitative mass spectrometry will be performed to uncover differentially expressed transcripts and proteins, respectively, to identify pathways critical for TMPRSS13-mediated functions. In Aim 2, we will develop and test new inhibitors of TMPRSS13 in breast cancer cellular models. Medicinal chemistry will be used to identify TMPRSS13 inhibitors in our collection of serine protease peptidomimetic compounds that inhibit TMPRSS13 activity. Compounds will be modified at each position to increase potency, selectivity, and stability. TMPRSS13 inhibitors will be validated in 2D and 3D cell culture models of breast cancer using quantitative proliferation/survival/apoptosis, and invasion assays to assess anticancer effects. In Aim 3, we will evaluate the efficacy of novel TMPRSS13 inhibitors in vivo using patient derived xenograft (PDX) models. Conventional chemotherapeutics often cause severe side effects and carry a significant risk of developing resistance. Preliminary data indicate that TMPRSS13 targeting leads to increased chemosensitivity in breast cancer cells. Therefore, the therapeutic effect of TMPRSS13 inhibition as mono-therapy as well as combination-therapy with standard of care chemotherapy drugs in vivo will be evaluated. The combination of state-of-the art mouse models and 2D and 3D cell culture based assays with transcriptome and proteome profiling and drug discovery encompasses an innovative and impactful strategy for studying human cancerous disease.